David Busbee - US grants

This research proposes to examine the binding of lipophilic polynuclear aromatic hydrocarbon carcinogens to serum lipoproteins (LP), correlating such binding to the component composition of the lipoproteins. Data show PAH carcinogens bound to LP enter cells potentiated level are subsequently to cellular DNA. We will investigate the LP mediated internalization of PAH carcinogens in vitro. Baboon smooth muscle cells, human lymphocytes, normal and LDL receptor deficient human fibroblast cell lines, and the rodent cell lines C3H/10T1/2 clone 8 and V-79 will be employed in this study. We propose to examine receptor mediated LP entry into cells as a route of PAH entry. We will assess LP stabilization of sequestered PAH carcinogens prior to and during cell entry of the LP from an aqueous environment. We will use V-79 to assess LP mediated PAH entry into cells and interaction with cellular DNA to produce detectable mutants and sister chromatid exchange. We will use differential transformation of 10T1/2 cells in the presence of PAH carcinogens and varying LP types and concentrations. We will use the smooth muscle cells (baboon embryonic) to assess LP mediated carcinogen transport into cells with subsequent metabolism of the carcinogens to reactive forms capable of binding cellular DNA. These studies will provide several levels of information related to the LP and LP-potentiated entry of carcinogen into cells. We will be able to examine the PAH entry, metabolism, binding to DNA, initiation of mutations, initiation of chromosomal defects, and initiation of transformation. We will also assess the initiation of DNA repair in these various cells exposed to different types and concentrations of LP. We will assess why human lipoproteins bind different levels of PAH carcinogens, and if the component composition of the lipoprotein dictates the PAH binding characteristics. This research crosses lines of disciplines between genetics, molecular biology, biochemistry, and clinical studies of atherogenesis and carcinogenesis. The interaction of LP with carcinogens in the production of human disease states dictates the scope of the research. We wish to understand how lipoproteins and lipophilic carcinogens interact in the currently known synergistic manner in the production of human disease.

This research proposes to examine the relationship between aging and decreased DNA synthesis associated with DNA excision repair and with mitogen-stimulated blastogenesis in human and mouse cells. Data from an ongoing research program show that DNA pol-a is present in quiescent cells in an inactive form which is activated upon stimulation of the cells to initiate either cell division or DNA excision repair. Data show that inactive DNA pol-a does not bind DNA template/primer, and that initiation of DNA synthesis necessary for either DNA excision repair or mitogen-stimulated cell division does not proceed in the absence of DNA pol-a activation. Inactive DNA pol-a which is treated with phosphatidylinositol (PI), phosphatidylinositol kinase (PIK), and ATP, or phosphatidylinositol-4-monophosphate (PIP) alone, is activated by transfer of a phosphate from PIP to a subunit of the enzyme. Phosphorylated DNA pol-a has a high binding affinity for DNA template/primer, and initiates DNA synthesis. Active DNA pol-a may be inactivated by treatment with alkaline phosphatase. Fibroblasts treated with the carcinogen r-7,t-8-dihydroxy-t-9,10- epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE) begin unscheduled DNA synthesis correlated with phosphorylation of DNA pol-a and an increase in specific activity of DNA pol-a. Lymphocytes treated with the mitogen phytohemagglutinin initiate cell division correlated with phosphorylation of DNA pol-a and an increase in specific activity of the enzyme. Activation of DNA pol-a is directly associated with phosphatidylinositol mobilization in stimulated cells, and fibroblasts or lymphocytes deprived of phospholipids do not activate DNA pol-a efficiently. Our data show that both unscheduled DNA synthesis in carcinogen-treated fibroblasts, and blastogenesis in mitogen- stimulated lymphocytes, decrease as a function of increased age in human subjects, and that decreased DNA synthesis correlates with decreased specific activity of DNA pol-a isolated from the cells. We propose to examine the efficiency of DNA pol-a activation in human lymphocytes, in human and mouse fibroblasts, and in mouse hepatocytes as a function of age. We have characterized the primary phosphorylation mechanism which activates DNA pol-a. We will determine the mechanism(s) involved in regulation of that phosphorylation cascade. We will determine the effects of aging in human and mouse cells on both the phosphorylation cascade and the mechanisms of activation of the cascade. We will determine the difference in DNA pol-a activation by phosphorylation in early and late passage fibroblasts from young and old mice and humans, in hepatocytes from young and old mice, and in lymphocytes from young, middle aged, and older humans.

We propose to establish the expression of an adult form of DNA polymerase alpha as a biomarker of aging in rodent model systems, and to determine whether dietary restriction can alter the age-related progressive expression of the adult form of this enzyme. Data from an ongoing research project show that DNA polymerase alpha isolated from human cells in vitro, or from mouse tumor cells grown in vitro or as a solid tumor, have two forms of DNA polymerase alpha. One enzyme form is indistinguishable from polymerase alpha purified from fetal- derived fibroblasts. This isozyme, which is highly active, declines as a present of total polymerase protein with increased age of the cell donor. A second isozyme, which has low activity but can be transiently activated by interaction with a product of the phosphoinositide cascade, increases as a percent of total polymerase protein with increased age of the cell donor. These isozymes of DNA polymerase alpha purified from either human or mouse cells can be separated because they differ in charge, in their affinity of binding to an anti-DNA polymerase alpha monoclonal IgG specific to what we call the polymerase regulatory subunit, and in their affinity of binding to enzymatically gapped DNA template/primer. The fetal-type isozyme of DNA polymerase alpha exhibits about a 10-fold greater specific activity than is shown by adult-type enzyme, and can not be further activated. The adult-type isozyme of DNA polymerase alpha has low specific activity, but can be transiently activated by interaction with inositol-1,4-bisphosphate, a hydrolysis product of phosphatidylinositol-4-monophosphate. Activated adult-type polymerase alpha can not be distinguished from the fetal form of the enzyme. The progressive increase in expression of adult-type DNA polymerase alpha with increased age of the cell donor is quite significant, with fetal cells showing 0% of the adult enzyme form, and cells derived from a 66 year old male exhibiting 94% of the total polymerase protein as adult-type enzyme. We will characterize the expression of DNA polymerase alpha fetal vs adult forms as a function of age in mouse and rat model systems, and in the F1 hybrids of the respective rat and mouse strains. We will determine whether dietary restriction alters the progressive decline of fetal enzyme expression and the progressive increase in adult enzyme expression as a function of increased age of the animals. We will compare DNA polymerase alpha expression as an aging biomarker in the rodent systems with DNA polymerase alpha expression as an aging biomarker in human fibroblasts to assess the potential for future use of this enzyme system in human biomarker studies.

biomedical equipment resource; biomedical equipment purchase;